Method for measuring flourescence, apparatus for measuring flourescence and apparatus for evaluating sample using it

ABSTRACT

A sample S is irradiated with pulsed pumping light supplied from a pumping light source  1 , fluorescence generated by the sample S is detected by a photodetector  5  by way of a condensing optical system  3  and a spectroscope  4,  and the fluorescence time waveform is subjected to a data analysis in a data processing unit  62  in a controller  6,  so as to compute waveform data and physical quantities such as fluorescence lifetime. Here, the pumping light time waveform is fixedly arranged with respect to a time axis used for the data analysis, a plurality of fitting calculations are carried out while moving the fluorescence time waveform and fitting range from an initial position earlier than a pumping light peak to a later end position, and optimal measurement waveform data is selected according to a predetermined selection criterion. This realizes fluorescence measuring method and apparatus which can compute waveform data and the like accurately and efficiently regardless of fluctuations in the fluorescence time waveform, and a sample evaluating apparatus using the same.

TECHNICAL FIELD

[0001] The present invention relates to fluorescence measuring methodand apparatus for measuring the time waveform of fluorescence releasedfrom a sample pumped with pumping light and carrying out a data analysisso as to compute waveform data and the like, and a sample evaluatingapparatus using the same.

BACKGROUND ART

[0002] In a fluorescence measuring apparatus which measures, in atime-resolved manner, fluorescence generated in a sample, so as toacquire information such as fluorescence lifetime, the sample is pumpedupon irradiation with pulsed pumping light from a pumping light source,and the fluorescence generated by and emitted from the pumped sample,the change in its intensity along with a lapse of time in particular, isdetected by a photodetector such as photomultiplier. Then, the timewaveform of fluorescence obtained by the fluorescence detection dataoutputted from the photodetector is subjected to a data analysis such asarithmetic operation executed in a data processing unit, so as tocompute waveform data, fluorescence lifetime, and the like.

[0003] The time waveform of fluorescence obtained in such a fluorescencemeasuring apparatus becomes a fluorescence decay time waveform in whichthe intensity of fluorescence released after irradiation with pumpinglight pulses decays with time. In practice, however, the fluorescencetime waveform is measured in a state where the exponential fluorescencedecay time waveform derived from a fluorescent component and the timewaveform of pumping light caused by the time waveform of apparatusresponse such as the finite pulse width of pumping light resulting fromthe apparatus are convoluted with each other.

[0004] Therefore, when computing waveform data, fluorescence lifetime,and the like by carrying out a data analysis, the fluorescence decay andapparatus response time waveforms are deconvoluted from each other byusing the pumping light time waveform measured separately from themeasured fluorescence time waveform. At the same time, the fluorescencedecay time waveform is subjected to a fitting calculation employing afunction system such as exponential function, so as to compute waveformdata for specifying the time waveform and physical quantities such asfluorescence lifetime (see, for example, Japanese Patent Publication No.2911167).

DISCLOSURE OF THE INVENTION

[0005] The respective time waveforms of fluorescence and pumping lightemployed for the above-mentioned data analysis including the fittingcalculation are normally measured by using the same measuring system,i.e., the same optical system, photodetector, and the like, in order toprevent the time waveforms from being affected by differences in theconfiguration of measuring systems. Here, these time waveforms aredetermined by measuring the fluorescence and pumping light at respectivetimes different from each other. When separate measurement operationsare carried out by using the same measuring system as such, thefluctuation in time waveform occurring due to the drift in operatingstates of the measuring apparatus along with the lapse of timetherebetween may become a problem in terms of data analysis.

[0006] Recently, in particular, fluorescence measuring apparatus foracquiring information such as fluorescence lifetime have been in theprocess of being applied to various fields of sample evaluations such ascrystal quality evaluation for semiconductor wafers. Also, as theapplication field for such a fluorescence measuring apparatus expands,there has been occurring a necessity to carry out a number offluorometric operations continuously in a time as short as possible, forexample, so as to evaluate a plurality of parts on a semiconductor waferby fluorometry.

[0007] Here, measurement conditions in each fluorometric operationchange with time due to such phenomena as the change in operating stateof the measuring apparatus caused by heat and the like, and thefluctuation in oscillation timing of a pulse laser light source employedas a pumping light source from the timing of clock signals forsynchronization with the photodetector. Therefore, if the respectivefluorescence time waveforms obtained at separate fluorometric operationsare used as they are, the position of fluorescence time waveform on thetime axis may shift from the pumping light time waveform determined by aseparate measurement operation beforehand among the fluorometricoperations.

[0008] As the measured fluorescence time waveform thus shifts on thetime axis of data analysis and from the pumping light time waveformfixed with respect to the time axis, data analyses such as fittingcalculation by deconvolution may not be carried out correctly due to theshift in time, whereby the waveform data and fluorescence lifetimecannot be computed accurately. If the pumping light measurement is to becarried out each time when fluorometry is conducted, the time requiredfor fluorometry and sample evaluation may become longer while theinfluence of shift of time waveform is suppressed.

[0009] In view of the foregoing problems, it is an object of the presentinvention to provide fluorescence measuring method and apparatus whichcan compute waveform data and the like correctly and efficientlyregardless of fluctuations in fluorescence time waveforms, and a sampleevaluating apparatus using the same.

[0010] For achieving such an object, the fluorescence measuring methodof the present invention comprises (1) a pumping step of irradiating asample with pulsed pumping light; (2) a light-detecting step ofdetecting fluorescence released from the sample pumped with the pumpinglight; and (3) a data processing step of subjecting a time waveform offluorescence detected by the light-detecting step to a data analysisincluding a fitting calculation in a fitting range acting as apredetermined time range fixedly set for the time waveform offluorescence so as to compute waveform data; (4) wherein, in the dataprocessing step, a time waveform of pumping light determined beforehandand the time waveform of fluorescence are arranged on a time axis usedfor the fitting calculation such that a fluorescence peak of the timewaveform of fluorescence is placed at an initial position earlier orlater by a predetermined time width than a time position substantiallycoinciding with a pumping light peak of the time waveform of pumpinglight, and then, while moving the time waveform of fluorescence andfitting range or the time waveform of pumping light with respect to thetime axis to an end position on the time axis on the opposite side ofthe time position where the fluorescence peak substantially coincideswith the pumping light peak from the initial position, respectivefitting calculations are carried out at a plurality of time positionsdifferent from each other with reference to the time waveform of pumpinglight, and a waveform data item selected according to a predeterminedselection criterion from a plurality of waveform data items respectivelycomputed in the fitting calculations is employed as final measurementwaveform data.

[0011] The fluorescence measuring apparatus of the present inventioncomprises (a) pumping means for irradiating a sample with pulsed pumpinglight; (b) light-detecting means for detecting fluorescence releasedfrom the sample pumped with the pumping light; and (c) data processingmeans for subjecting a time waveform of fluorescence detected by thelight-detecting means to a data analysis including a fitting calculationin a fitting range acting as a predetermined time range fixedly set forthe time waveform of fluorescence so as to compute waveform data; (d)wherein the data processing means arranges a time waveform of pumpinglight determined beforehand and the time waveform of fluorescence on atime axis used for the fitting calculation such that a fluorescence peakof the time waveform of fluorescence is placed at an initial positionearlier or later by a predetermined time width than a time positionsubstantially coinciding with a pumping light peak of the time waveformof pumping light, and then, while moving the time waveform offluorescence and fitting range or the time waveform of pumping lightwith respect to the time axis to an end position on the time axis on theopposite side of the time position where the fluorescence peaksubstantially coincides with the pumping light peak from the initialposition, carries out respective fitting calculations at a plurality oftime positions different from each other with reference to the timewaveform of pumping light, and employs a waveform data item selectedaccording to a predetermined selection criterion from a plurality ofwaveform data items respectively computed in the fitting calculations asfinal measurement waveform data.

[0012] In the above-mentioned fluorescence measuring method andapparatus, a fluorescence time waveform and a pumping light timewaveform measured separately from fluorometry are respectively arrangedat predetermined time positions on the time axis for a fittingcalculation. Then, the fluorescence time waveform or pumping light timewaveform is moved from the initial position where the fluorescence peakis located earlier or later by a predetermined time width than thepumping light peak, to the end position on the opposite side (from theearlier initial position to the later end position or from the laterinitial position to the earlier end position).

[0013] If there is no shift in thus measured fluorescence time waveformin the time-axis direction here, so that the respective positions (timepositions) of fluorescence and pumping light time waveforms on the timeaxis coincide with each other, the fluorescence peak in the fluorescencetime waveform will be located at the time position of the pumping lightpeak or later. Therefore, if the initial and end positions forspecifying the range for mutually moving the fluorescence and pumpinglight time waveforms in a data analysis are appropriately set on thetime axis, the above-mentioned fluorescence time waveform position atthe time when there is no shift will always be included in the movingrange.

[0014] In the fluorescence measuring method and apparatus in accordancewith the present invention, by contrast, a plurality of fittingcalculations are executed while moving the time waveform of fluorescenceor pumping light on the time axis as mentioned above, so as to determinea plurality of waveform data items from a single fluorescence timewaveform, and an optimal measurement waveform data item is selected froma plurality of waveform data items while using as a selection criterionan amount to become a determination criterion for determining whetherthe result of fitting calculation is favorable or not or the like. Thismakes it possible to compute waveform data, individual physicalquantities, and the like efficiently with a sufficient accuracyregardless of whether the fluorescence time waveform shifts or not.

[0015] In the data analysis, the time range (fitting range) forspecifying the data range for carrying out fitting is set to a fixedtime position with respect to the fluorescence time waveform instead ofthe time axis. When moving the fluorescence time waveform, the fittingrange is moved on the time axis together with the fluorescence timewaveform, so as to execute respective fitting calculations. As aconsequence, each of a plurality of fitting calculations executed isalways carried out under an optimal condition.

[0016] Preferably, in the plurality of fitting calculations within themoving range of fluorescence or pumping light time waveform, the movinginterval on the time axis for executing the fitting calculations and thelike are appropriately set according to the numerical accuracy requiredfor waveform data and physical quantities and the like. As for theinitial and end positions, the initial position is specified accordingto the time width from the pumping light peak and the like. The endposition may be set according to the time width from the pumping lightpeak as with the initial position, or determination based on theselection criterion may be carried out each time the fitting calculationis executed and the time position where the waveform data to be selectedis determined may be used as the end position so as to terminate thedata analysis.

[0017] The sample evaluating apparatus in accordance with the presentinvention comprises the above-mentioned fluorescence measuringapparatus, and sample evaluating means for evaluating the sample bycomparing the measurement waveform data obtained by the data processingmeans of the fluorescence measuring apparatus and reference waveformdata determined beforehand with each other.

[0018] This realizes a sample evaluating apparatus which can evaluate asample accurately and efficiently even when the measured fluorescencetime waveform shifts on the time axis. In apparatus for evaluatingsemiconductor wafers and the like, in particular, a number offluorometric operations are repeatedly executed for individual parts ofa sample (semiconductor wafer). Even in such a case, employing theabove-mentioned fluorescence measuring apparatus can minimize theinfluence of shift in the fluorescence time waveform caused byfluctuations in measurement conditions occurring between the individualfluorometric operations.

[0019] Thus configured sample evaluating apparatus can be employed notonly for the above-mentioned semiconductor wafer quality evaluation, butalso for various sample evaluations such as mass-screening in drugdevelopments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a diagram showing an embodiment of fluorescencemeasuring apparatus;

[0021]FIG. 2 is a graph showing an example of pumping light andfluorescence time waveforms;

[0022]FIG. 3 is a graph showing another example of pumping light andfluorescence time waveforms;

[0023]FIGS. 4A to 4D are schematic charts for explaining a data analysismethod in the fluorescence measuring apparatus shown in FIG. 1;

[0024]FIG. 5 is a schematic chart for showing how to select ameasurement waveform data item from a plurality of waveform data items;

[0025]FIG. 6 is a flowchart showing an example of fluorescence measuringmethod in the fluorescence measuring apparatus shown in FIG. 1;

[0026]FIG. 7 is a diagram showing an embodiment of sample evaluatingapparatus; and

[0027]FIG. 8 is a flowchart showing an example of sample evaluatingmethod in the sample evaluating apparatus shown in FIG. 7.

BEST MODES FOR CARRYING OUT THE INVENTION

[0028] In the following, preferred embodiments of fluorescence measuringmethod and apparatus in accordance with the present invention, and asample evaluating apparatus using the same will be explained in detailwith reference to the drawings. In the explanation of the drawings,constituents identical to each other will be referred to with numeralsidentical to each other without repeating their overlappingdescriptions. Ratios of dimensions in the drawings do not always matchthose explained.

[0029]FIG. 1 is a diagram showing an embodiment of the fluorescencemeasuring apparatus in accordance with the present invention. Thisfluorescence measuring apparatus comprises a pumping light source 1 forirradiating a sample S held by a sample holding means 2 with pumpinglight, a photodetector 5 for detecting fluorescence released from thesample S pumped with the pumping light, and a controller 6 for drivingand controlling the pumping light source 1 and the photodetector 5. InFIG. 1, the sample S in a gaseous form or the like held within a samplecell placed as the sample holding means 2 is shown as an example of thesample S.

[0030] As the pumping light source 1, a pulse light source which cansupply pulsed light having a predetermined wavelength and time width aspumping light is used. Employed as the photodetector 5 is one such asphotomultiplier (PMT) or streak camera, for example, which is capable oftime-resolved measurement for obtaining time waveforms.

[0031] Placed between the sample holding means 2 holding the sample Sand the photodetector 5 are a condensing optical system 3 and aspectroscope 4. The condensing optical system 3 has necessary opticalelements such as lenses, and condenses and converges onto thephotodetector 5 the fluorescence generated in pumped parts within thesample S pumped with the pumping light emitted from the pumping lightsource 1 and released into various directions.

[0032] The spectroscope 4 is placed as wavelength selecting means forselecting an optical component in a predetermined wavelength region inthe optical components detected by the photodetector 5 and making thusselected optical component incident on the photodetector 5. As suchwavelength selecting means, not only a spectroscope but also awavelength selecting filter or the like can be used. The wavelengthregion of light to be selected by wavelength selecting means such as thespectroscope 4 or wavelength selecting filter is appropriately set orchanged to a wavelength region including the wavelength of fluorescenceor pumping light to be detected by the photodetector 5.

[0033] The controller 6 for controlling operations of individual partsof the fluorescence measuring apparatus and the like has a drivingcontrol unit 61, a data processing unit 62, and a range setting unit 63.Employable as such a controller 6 is a personal computer (PC), forexample, which invokes control software, data processing software, andthe like, thereby realizing individual functions necessary as thecontroller 6.

[0034] The pumping light source 1, the spectroscope 4, and thephotodetector 5 are driven by clock signals and other control signalssupplied from the driving control unit 61 of the controller 6. As aconsequence, with respect to the pulsed pumping light supplied from thepumping light source 1 at predetermined clock time intervals, thefluorescence released from the sample S upon irradiation with theindividual pumping light pulses and its change with time are detected bythe photodetector 5. The fluorescence detection data based on thusdetected fluorescence is fed into the controller 6 according to thedetection signals.

[0035] According to data analysis instructions based on clock signalsand the like from the driving control unit 61, the data processing unit62 executes data processing such as necessary signal processing and dataanalysis with respect to the detection signals inputted from thephotodetector 5. Initially, in the data analysis with respect to thefluorescence detection data, the fluorescence time waveform indicativeof the temporal change in the fluorescence intensity is determined forthe fluorescence released from the sample S and detected by thephotodetector 5 due to pumping light pulses corresponding thereto.

[0036] Thus obtained fluorescence time waveform is subjected to a dataanalysis including a fitting calculation in which the time waveform isfitted with a predetermined curve (function system) or the like, so asto compute waveform data, necessary physical quantities, and the like.Examples of thus computed waveform data and physical quantities includephysical quantities such as individual parameters for determining thecurved form of time waveform, fluorescence lifetime of fluorescentcomponents, and fluorescence intensity. The range setting unit 63 setsthe time range of fitting (hereinafter referred to as fitting range)used for fitting calculations in the data processing unit 62. A specificdata analysis method, setting of the fitting range, and the like will beexplained later.

[0037] Preferably, a display 7 (see FIG. 1) for displaying thefluorescence time waveform obtained by the data processing unit 62, thecomputed waveform data, the fluorescence lifetime, and the like isconnected to the controller 6 as necessary.

[0038] When pulsed pumping light is supplied from the pumping lightsource 1 and irradiates the sample S (pumping step) in the foregoingconfiguration, thus pumped sample S releases fluorescence, which is thendetected by the photodetector 5 by way of the condensing optical system3 and spectroscope 4 (light-detecting step). Thereafter, in the dataprocessing unit 62 of the controller 6, a data analysis is carried outfor thus measured fluorescence time waveform, so as to compute waveformdata and the like (data processing step).

[0039] A fluorescence measuring method in the fluorescence measuringapparatus shown in FIG. 1 will now specifically be explained whileillustrating its data analysis method (data processing method).

[0040] First, the fluorescence time waveform obtained by thefluorescence measurement after the pumping light pulse irradiation willbe explained.

[0041]FIG. 2 is a graph showing an example of the time waveform ofpumping light emitted from the pumping light source 1 to the sample Sand the time waveform of fluorescence released from the sample S afterthe pumping light pulse irradiation. In this graph, the abscissaindicates the time axis (ns) used for the data analysis, whereas theordinate indicates the detected number (counts; detected intensity) ofpumping light or fluorescence. Here, in conformity to the fact that thetime waveform of fluorescence decay is exponential, the detected numberin the ordinate corresponding to the fluorescence intensity is indicatedby log scale. The time axis on the abscissa corresponds to the lapse oftime at the time of measuring fluorescence or pumping light.

[0042] Here, the pumping light time waveform shown in FIG. 2 in additionto the fluorescence time waveform is one to be referred to in the dataanalysis of fluorescence time waveform as will be explained later. Forpreventing differences in the configuration of measuring systems fromaffecting time waveforms, the pumping light time waveform is normallymeasured by using the same measuring system, i.e., the same opticalsystem, photodetector, and the like, used for fluorometry.

[0043] In the configuration shown in FIG. 1, pumping light can bemeasured by the photodetector 5 by way of the condensing optical system3 and spectroscope 4, for example, when the sample holding means 2 holdsa scatterer, which generates no fluorescence, in place of the sample Sto be subjected to fluorometry. Alternatively, a separate optical systemfor guiding a part of pumping light to the photodetector may beprovided. As for the wavelength selection effected by the spectroscope 4(or a wavelength selecting filter and the like), since the pumping lightand fluorescence have respective wavelengths different from each other,it is necessary that the pumping light measurement be carried out whilechanging the wavelength region to be selected.

[0044] When a sample is pumped with pulsed pumping light, thefluorescence intensity of fluorescence released from the sampleindicates the temporal change of fluorescence decaying with time afterthe pumping light irradiation. The fluorescence time waveform caused bythe change in time becomes an exponential fluorescence decay timewaveform in the case where the time width of pumping light pulse isnegligible. Then, from the amplitude and attenuation factor of theexponential decay curve, the fluorescence intensity and fluorescencelifetime of the fluorescence pumped in the sample can be calculated.

[0045] On the other hand, the fluorescence time waveform actuallymeasured in the fluorescence measuring apparatus is one deformed from anideal decay curve to some extent due to influences caused by apparatusresponse. Namely, the fluorescence time waveform is obtained in a statewhere the above-mentioned exponential fluorescence decay time waveformresulting from the temporal change of fluorescence itself is convolutedwith the pumping light time waveform determined by the finite pulse timewidth of pumping light, the shift in time depending on the optical path,and the like.

[0046] In the example of time waveforms shown in FIG. 2, the pumpinglight time waveform E indicates the pumping light pulse having a pulsetime width of about several nanoseconds which cannot be neglected. Bycontrast, the fluorescence time waveform F is convoluted with such apumping light time waveform E, whereby its detected number risestogether with the starting of pumping light supply, and increases untilthe fluorescence intensity reaches a peak at a certain point in time(hereinafter referred to as fluorescence peak), after which thefluorescence intensity decays substantially exponentially (linearly inthe log scale). Here, the time position of the fluorescence peak on thetime axis is located at the time position of the pumping light intensitypeak (hereinafter referred to as pumping light peak) in the pumpinglight time waveform or later as shown in FIG. 2.

[0047] A data analysis method for computing the waveform data,fluorescence lifetime, and the like from the fluorescence time waveformwill now be explained.

[0048] In order to acquire information such as waveform data andfluorescence lifetime by carrying out a data analysis with respect tothe fluorescence time waveform F convoluted with the pumping light timewaveform E as mentioned above, it is necessary that a data analysisincluding a fitting calculation by deconvolution be carried out by usingthe pumping light time waveform E and a fluorescence decay time waveformassumed by appropriate function system (e.g., exponential function) andparameters (e.g., amplitude and attenuation factor).

[0049] In this data analysis, a fitting calculation (deconvolutionoperation) isolates the component of pumping light time waveform Econvoluted with the fluorescence time waveform F, so as to extract thefluorescence decay time waveform. Then, according to the result offitting calculation with respect to the fluorescence decay timewaveform, not only waveform data such as amplitude and attenuationfactor, but also physical quantities such as fluorescence intensity andfluorescence lifetime are computed.

[0050] The above-mentioned fitting calculation with respect to thefluorescence time waveform F is executed while setting on the time axisa fitting range which is a time range for specifying detection data usedfor the fitting calculation in the measured fluorescence time waveformF. As an example of such a fitting range, FIG. 2 shows a time range Rtset with respect to the fluorescence time waveform F.

[0051] Here, the operating state of the fluorescence measuring apparatusmay drift during the measurement along with the lapse of time from themeasurement of the pumping light time waveform used for deconvolution tothe fluorescence measurement or the lapse of time while executing aplurality of fluorometric operations to be carried out repeatedly. Inthis case, the fluctuation of fluorescence time waveform caused by thechange in apparatus state between the individual fluorometric operationsmay become problematic in terms of data analysis.

[0052] When a pulse laser light source is used as the pumping lightsource 1, for example, its oscillation timing may vary within a certainlevel of time range with respect to clock signals for synchronousdriving supplied from the driving control unit 61 of the controller 6.In this case, the pumping light irradiation timing for the sample Sshifts from the clock signal timing, whereby the time position of timewaveform on the time axis shifts with respect to the fluorescencegenerated upon the pumping light pulse irradiation as well.

[0053]FIG. 3 shows the pumping light and fluorescence time waveforms inthe case where such a fluorescence time waveform shift is generated. Thepumping light time waveform E is arranged at a fixed time position withrespect to the time axis used for a fitting calculation so as to belocated at a time position corresponding to the fluorescence timewaveform F (see FIG. 2) in the case with no time waveform shift in thedata analysis of fluorescence time waveform F carried out for eachfluorometric operation. Here, if the fluorescence time waveform F ismeasured while being shifted to the earlier or later side on the timeaxis as shown in FIG. 3, the pumping light time waveform E and thefluorescence time waveform F will deviate from each other on the timeaxis, whereby the deconvolution operation cannot be carried outcorrectly. Also, it will be problematic in terms of executing thefitting calculation if the fitting range Rt is set to a time range fixedwith respect to the time axis, since the fitting range Rt and thefluorescence time waveform F similarly deviate from each other.

[0054] By contrast, the fluorescence measuring apparatus of theembodiment shown in FIG. 1 and the fluorescence measuring method carriedout thereby sequentially execute a plurality of fitting calculations fora data analysis at respective time positions different from each otherwhile moving both of the fluorescence time waveform F and fitting rangeRt with respect to the time axis. Then, from a plurality of waveformdata items respectively computed in the fitting calculations, an optimalwaveform data item is selected as final measurement waveform data, so asto realize a data analysis minimizing the influence of fluctuations intime waveforms in individual fluorometric operations such as theabove-mentioned shift in fluorescence time waveform F.

[0055]FIGS. 4A to 4D are schematic charts for explaining the dataanalysis method in the fluorescence measuring method using thefluorescence measuring apparatus shown in FIG. 1. Here, in each of theschematic graphs shown in FIGS. 4A to 4D, the abscissa indicates thetime axis t of the data analysis (corresponding to the lapse of time inmeasurement), whereas the ordinate indicates the detected numbers ofpumping light and fluorescence (corresponding to the pumping lightintensity and fluorescence intensity) by log scale.

[0056] In each of FIGS. 4A to 4D, for simplifying the explanation andillustration, the pumping light time waveform E is indicated by a pulsedwaveform neglecting the pulse time width whereas the fluorescence timewaveform F is indicated by a triangular waveform whose rise and decayeach become linear in log scale. As for the pumping light waveform E,the time position where the pumping light peak is fixed on the time axisis defined as t0.

[0057]FIG. 4A shows the state where the fluorescence time waveform F ismeasured as being shifted to the “+” direction (positive direction) ofthe time axis with respect to the time axis used for the fittingcalculation and the pumping light time waveform E fixedly arranged withrespect to the time axis. If the fitting range used for the fittingcalculation in the data analysis is set to a time range R0 (dotted line)fixed with respect to the time axis in this case, the fluorescence timewaveform F will deviate from the fitting range R0 as the time waveformshifts, whereby correct waveform data and physical quantities cannot beobtained as results of the fitting calculations.

[0058] In the fluorescence measuring method of this embodiment, bycontrast, a fitting range is set as a time range fixed with respect tothe fluorescence time waveform F instead of the time axis, as with thetime range Rt (solid line) shown in FIG. 4A. The fitting range Rt is setautomatically or manually by an operator (range setting step) by usingthe fluorescence time waveform in the initial fluorometric operation ora preliminary fluorometric operation carried out prior to the actualmeasurement in the range setting unit 63 of the controller 6 (see FIG.1).

[0059] Next, the fluorescence time waveform F measured at the timeposition shifted from the pumping light time waveform E as shown in FIG.4A is moved until the respective time positions of the pumping light andfluorescence peaks substantially coincide with each other (FIG. 4B).Then, the fluorescence time waveform F is further moved and arrangedsuch that the fluorescence peak is positioned at an initial position t1which is earlier by a predetermined time width than the time position t0substantially coinciding with the pumping light peak (t1<t0) (FIG. 4C).

[0060] The above-mentioned initial position t1 is the time position tobecome the start position for a plurality of fitting calculations to beexecuted in the data analysis. Then, the fluorescence time waveform F ismoved from the initial position t1 earlier than the pumping light peaktime position t0 to the end position t2 later than the time position t0(t2>t0) (FIG. 4D).

[0061] Here, while the fluorescence time waveform F is sequentiallymoved from the initial position t1 in the “+” direction at predeterminedmoving time intervals, a fitting calculation is carried out at each ofthus moved time positions, so as to compute waveform data. In this case,since the fitting range Rt is set as a time range fixed with respect tothe fluorescence time waveform F as mentioned above, it is moved withrespect to the time axis together with the fluorescence time waveform F.

[0062] Among the respective waveform data items computed in a pluralityof fitting calculations executed at the individual time positions, oneselected as optimal according to a predetermined selection criterion isemployed as final measurement waveform data with respect to thefluorescence time waveform F. As the selection criterion for waveformdata, a quantity acting as a determination criterion for determiningwhether the result of a fitting calculation executed is favorable or notor the like is used, so as to select the best fitting calculationresult, thereby computing final measurement waveform data, and physicalquantities such as fluorescence lifetime.

[0063] According to the above-mentioned fluorescence measuring apparatusand method, if the initial position t1 and end position t2 specifyingthe range for moving the fluorescence time waveform at the time ofexecuting a plurality of fitting calculations are appropriately arrangedon the time axis, the time position of the fluorescence time waveform Fin the case where there is no shift in the time waveform will always beincluded within the moving range. While moving the fluorescence timewaveform F within the moving range, a plurality of fitting calculationsare executed, so as to determine a plurality of waveform data items froma single fluorescence time waveform F, and an optimal measurementwaveform data item is selected from a plurality of waveform data itemsaccording to a predetermined selection criterion. Here, regardless ofwhether a shift occurs in the fluorescence time waveform or not,waveform data, physical quantities such as fluorescence lifetime, andthe like can always be computed efficiently with a sufficient accuracyand numeric precision.

[0064] Also, the fitting range Rt employed for each of a plurality offitting calculations is set as a time range fixed to the fluorescencetime waveform F instead of the time axis and, while moving the fittingrange Rt as the fluorescence time waveform F moves, the fittingcalculation is carried out at each time position (see FIGS. 4C and 4D).As a consequence, each fitting calculation can be executed under afavorable condition.

[0065] Preferably, as for the movement of the fluorescence time waveformF from the initial position t1 to the end position t2, moving timeintervals on the time axis are appropriately set so as to yield anumerical accuracy required for quantities to be determined such aswaveform data and fluorescence lifetime, and the fitting calculation isexecuted at each of the time positions moved at the moving intervals.Setting the moving time intervals as such can improve the accuracy ofeach value computed and the efficiency of the data analysis includingthe fitting calculation at the same time. As a specific example ofmoving time interval setting methods, if time waveform data is digitizedinto a plurality of channels, the fitting calculation may be executedeach time when the fluorescence time waveform F is moved by 1 ch(channel) or a plurality of channels.

[0066] As for the initial position t1 and end position t2, the initialposition t1 is specified by the time width from the pumping light peaktime position t0 set beforehand. On the other hand, the end position t2may be set according to the time width or the like from the timeposition t0 as with the initial position t1, or a determination based onthe above-mentioned selection criterion may be carried out each time thefitting calculation is executed, and the data analysis maybe terminatedwhile defining as the end position the time position where the waveformdata to be selected is determined.

[0067] Preferably, the fitting range Rt employed for the fluorescencetime waveform F is set beforehand in the range setting unit 63 of thecontroller 6. As an example of specific method of setting a fittingrange in the range setting unit 63, it may automatically be set from thefluorescence time waveform obtained by the initial fluorometricoperation or a preliminary fluorometric operation. Alternatively, whilethis fluorescence time waveform is displayed on the display 7, the rangemay manually be set by an operator manipulating a mouse cursor or thelike.

[0068] A method of setting the fitting range Rt to a time range fixedwith respect to the fluorescence time waveform F is configured suchthat, while the fluorescence time waveform is specified by the timeposition of the fluorescence peak in the fluorescence time waveform F onthe time axis, the fitting range is set with reference to thisfluorescence peak. This can set respective favorable fitting ranges forthe fluorescence time waveforms obtained by individual measurementoperations.

[0069] As a specific fitting range, a time range is preferably set so asto exclude the rising and decaying end parts of the time waveforms (bothend parts of the time waveform), susceptible to statistical deviationsand noise, yielding a smaller detected number of fluorescence. Morespecifically, for example, there is a method in which the fluorescenceintensity of the fluorescence peak is employed as a referencefluorescence intensity, and the start and end points of the fittingrange are set by a predetermined range (e.g., at least 20%) in terms offluorescence intensity % with respect to the reference fluorescenceintensity. In another method, the position of the fluorescence peak onthe time axis is used as a reference time position, and the start andend points of the fitting range are set according to a predeterminedtime range determined with respect to the reference time position.

[0070] Here, such a fitting range may be set such that both of its startand end positions are located within the decay waveform later than thefluorescence peak, or such that the start point is within the risingwaveform earlier than the fluorescence peak whereas the end point iswithin the decay waveform later than the fluorescence peak.

[0071] As the selection criterion for selecting the final measurementwaveform data from the respective waveform data items computed by aplurality of fitting calculations, χ² values determined in the fittingcalculations are preferably used. This selecting method will beexplained with reference to the graph shown in FIG. 5. FIG. 5 is aschematic chart showing the change in respective χ² values obtained in aplurality of fitting calculations carried out from the initial positiont1 to the end position t2, in which the abscissa indicates the timeposition to which the fluorescence time waveform F has moved, whereasthe ordinate indicates the respective χ² values obtained by the fittingcalculations at individual time positions.

[0072] The χ² value (chi-square value) is a value determined as an indexfor determining whether the result of calculation of each fittingexecuted in an approximate calculation such as nonlinear least-squaresmethod used in such a fitting calculation is favorable or not, andbecomes a smaller value approximating 1 (χ²>1) as the fitting conditionis better. Namely, when fitting calculations are started from theinitial position t1 earlier than the time position t0 in theabove-mentioned data analysis method, the χ² value decreases as the timeposition of the fluorescence time waveform F moves in the “+” direction.Then, after being minimized at a certain time position tc at the pumpinglight peak time position or later, the χ² value increases again towardthe end position t2.

[0073] If the waveform data computed by the fitting calculation in whichthe χ² value is minimized, i.e., the waveform data computed by thefitting calculation at the time position tc in FIG. 5, is selected asthe final measurement waveform data, an optimal measurement waveformdata item can be chosen from a plurality of waveform data items. Whenthe χ² value, which is a value determined from a fitting calculation, isused as a determination criterion, a data analysis including waveformdata selection can automatically be executed in an efficient manner.

[0074]FIG. 6 is a flowchart showing an example of data analysis methodin the case where the χ² value obtained by the fitting calculation isused as a criterion for selecting waveform data.

[0075] Initially, in this data analysis method, a fitting range is setwith respect to the fluorescence time waveform F by a fluorescenceintensity % range with reference to the fluorescence peak, a time range,or the like (step S101). Next, the fluorescence time waveform F obtainedby measurement is moved on the time axis to the time position t0 wherethe fluorescence peak substantially coincides with the pumping lightpeak, and is further moved by a predetermined time width, in the “−”direction, whereby the fluorescence time waveform F is disposed at theinitial position t1 (S102). Then, at this initial position t1, the firstfitting calculation is executed while employing the above-mentionedfitting range (S103), so as to compute the first waveform data and χ²value.

[0076] Next, the execution of a plurality of fitting calculationsbetween the initial position t1 and the end position t2 is started.Namely, the fluorescence time waveform F is moved in the “+” directiontoward the end position t2 by a predetermined moving time interval(e.g., by +1 ch) (S104), and the next fitting calculation is executed atthus moved time position (S105). Next, the χ² value obtained by thisfitting calculation is compared with the χ² value obtained by theprevious fitting calculation (S106).

[0077] If the χ² value in this operation is not greater than that in theprevious operation, the χ² value has not reached the minimum value yet(see FIG. 5), whereby the moving of the fluorescence time waveform F(S104) and the fitting calculation (S105) are repeated. If the χ² valuein this operation is greater than that in the previous operation, the χ²value in the previous operation is the minimum value of the respectiveχ² values obtained in a plurality of fitting calculations. Therefore, inthis case, the plurality of fitting calculations are terminated, and thewaveform data computed in the fitting calculation at the time positionof fluorescence time waveform F in the previous operation (e.g. −1 ch)is selected as the final measurement waveform data (S107).

[0078] The foregoing data analysis method can compute waveform data andphysical quantities such as fluorescence lifetime with respect to thefluorescence time waveform obtained at each fluorometric operation whileattaining a sufficient accuracy and data analysis efficiency at the sametime regardless of whether a shift occurs in the fluorescence timewaveform or not.

[0079] The above-mentioned fluorescence measuring apparatus and methodcan be employed in sample evaluating apparatus for evaluating qualitiesand the like of various samples.

[0080] Recently, fluorescence measuring apparatus (fluorescence lifetimemeasuring apparatus) have been in the process of being applied toevaluations of semiconductor wafer crystal qualities and the like. Whenevaluating a semiconductor wafer, it is necessary that the distributionof crystal quality of the semiconductor wafer used in the product beevaluated beforehand within the wafer in order to improve the yield.When a fluorescence measuring apparatus is used for such a qualityevaluation, fluorescence lifetime and fluorescence intensity aredetermined by fluorometry using the semiconductor wafer to be evaluatedas a sample and are compared with those in a reference sample, so as toevaluate the crystal quality at each part of the semiconductor wafer.

[0081]FIG. 7 is a diagram showing an embodiment of sample evaluationapparatus using the fluorescence measuring apparatus in accordance withthe present invention. In this embodiment, the sample S to be evaluated(e.g., semiconductor wafer) is held in a state mounted on a sampleholding table 2 a acting as the sample holding means 2.

[0082] The pumping light source 1 is placed above the sample S held onthe sample holding table 2 a, whereas pumping light pulses supplied fromthe pumping light source 1 pass through a lens 11 and further through ahalf mirror 32 and a lens 31 which constitute the condensing opticalsystem 3, thereby irradiating a predetermined irradiating position onthe sample S which is a part to be evaluated in the sample S. Thefluorescence from the part on the sample S pumped with the pumping lightpulses is made incident on the photodetector 5 by way of the condensingoptical system 3 constituted by the lens 31, half mirror 32, variableoptical attenuator 33, and lens 34, and the spectroscope 4 (orwavelength selecting means such as a wavelength selecting filter). Here,the variable optical attenuator 33 is used with its optical attenuationbeing set as required for adjusting the light quantity when measuringpumping light or fluorescence.

[0083] The controller 6 has not only the driving control unit 61, dataprocessing unit 62, and range setting unit 63, but also a sampleevaluating unit 64 for evaluating a sample by referring to the result offluorometry. The functions, operations, and the like of the drivingcontrol unit 61, data processing unit 62, and range setting unit 63 aresimilar to those in the fluorescence measuring apparatus shown in FIG.1.

[0084] On the other hand, the sample evaluating unit 64 compares themeasurement waveform data obtained by the data analysis including aplurality of fitting calculations carried out in the data processingunit 62 with reference waveform data determined beforehand. Then, fromthe results of data comparison, the quality and the like of the part tobe evaluated in the sample S are evaluated.

[0085]FIG. 8 is a flowchart showing an example of sample evaluationmethod using the sample evaluating apparatus shown in FIG. 7. The sampleevaluating method shown in FIG. 8 illustrates one in which, while movingthe sample S in X-Y directions with the sample holding table 2 a actingas a movable table, individual parts on the sample S are sequentiallyirradiated with pumping light pulses from the pumping light source 1, soas to carry out evaluation by fluorometry as in the quality evaluationfor each part of a semiconductor wafer or the like.

[0086] First, in this sample evaluating method, a pumping light timewaveform used for a fitting calculation in a data analysis is carriedout (step S201). Here, since the wavelength of pumping light differsfrom that of fluorescence to be measured, the selecting wavelengthregion of the spectroscope 4 (or wavelength selecting filter) isswitched to the pumping light wavelength and set.

[0087] As mentioned above in regard to the fluorescence measuringapparatus of FIG. 1, the measurement of pumping light can be carried outwhen a scatterer generating no fluorescence is mounted on the sampleholding table 2 a in place of the sample S. At this time, the quantityof pumping light incident on the photodetector 5 can be adjusted by thevariable optical attenuator 33 placed in the condensing optical system3.

[0088] Next, while the wavelength selecting region of the spectroscope 4(or wavelength selecting filter) is switched to the fluorescencewavelength and set, a reference sample to become a reference for sampleevaluation is mounted on the sample holding table 2 a, and thefluorescence time waveform in the reference sample is measured (S202).If the fluorescence time waveform in the reference sample has alreadybeen measured and prepared, this time waveform data may directly be readout and used without carrying out fluorometry in the reference sample.

[0089] Once the fluorescence time waveform in the reference sample isobtained, a fitting range is set with reference to this time waveform(S203). In a specific example of method for setting a fitting range, thefluorescence time waveform in the reference sample is represented on thedisplay 7 connected to the controller 6, and an operator indicates thestart and end points of a fitting range by manipulating a mouse cursoror the like. According to thus indicated start and end points, the rangesetting unit 63 of the controller 6 sets a fitting range employed forthe actual measurement of fluorescence that follows. Specifically, afitting range is fixedly set with respect to the fluorescence timewaveform according to the intensity % range with respect to thefluorescence intensity at the fluorescence peak or the time range withrespect to the time position of the fluorescence peak.

[0090] Next, while employing thus set fitting range, the fluorescencetime waveform in the reference sample is subjected to a data analysisincluding a plurality of fitting calculations in the above-mentioneddata analysis method, so as to compute reference waveform data, and thefluorescence lifetime and fluorescence intensity derived therefrom(S204). If the fluorescence time waveform is not shifted, however, asingle fitting calculation may be sufficient for computing the referencewaveform data.

[0091] Once the reference waveform data is obtained, the measurementsample S to be evaluated is placed at a predetermined position on thesample holding table 2 a acting as a movable stage, and a sampleevaluation by fluorometry is started.

[0092] First, the sample holding table 2 a is driven, so as to move themeasurement sample S such that a predetermined part of the measurementsample S is irradiated with pumping light pulses from the pumping lightsource 1 (S205), and the fluorescence time waveform in the measurementsample S is measured while pumping light pulses are supplied from thepumping light source 1 (S206). Then, a plurality of fitting calculationsin the above-mentioned fitting range are employed, so as to subject thusobtained fluorescence time waveform to a data analysis, therebycomputing measurement waveform data, and the fluorescence lifetime andfluorescence intensity derived therefrom (S207).

[0093] Subsequently, in the sample evaluating unit 64, the measurementwaveform data computed for the measurement sample S is compared with thereference waveform data determined beforehand for the reference sample,so as to evaluate parts of the measurement sample S to be evaluated,thereby determining whether the quality is favorable or not and so forth(S208). As for the comparison between the measurement waveform data andreference waveform data, the waveform data themselves may be comparedwith each other, or they may be compared with each other in terms offluorescence lifetime and fluorescence intensity values.

[0094] After the sample evaluation is completed, it is investigatedwhether or not the fluorometry and sample evaluation have been executedfor all the evaluating parts of the measurement sample S needed to beevaluated (S209). If they have been executed, all of the fluorometry andsample evaluation for the measurement sample S are terminated. If thereare evaluating parts for which the measurement and evaluation have notbeen executed yet, the measurement sample S is further moved (S205), soas to execute the measurement, computation, and evaluation (S206, S207,S208) repeatedly.

[0095] Such a sample evaluating apparatus realizes one which canevaluate a sample accurately and efficiently even when a shift occurs inthe fluorescence time waveform. In a semiconductor wafer evaluatingapparatus or the like, in particular, a plurality of fluorometricoperations are repeatedly executed for individual parts of a sample(semiconductor wafer). When the above-mentioned fluorescence measuringapparatus is employed, influences of time waveform shifts occurringbetween individual fluorometric operations can be minimized in such acase as well.

[0096] Namely, for reducing influences of shifts in fluorescence timewaveform along with the lapse of time, the pumping light time waveformcan be measured before each of a plurality of fluorometric operationsexecuted. If the pumping light is measured each time as such, the timerequired for sample evaluation will become longer. In particular, forevaluating the quality of a semiconductor wafer or the like in moredetail, an automatic evaluation at a high resolution with a highthroughput is required, which necessitates a quite large number ofevaluating parts in the whole surface of the semiconductor wafer,thereby increasing the measurement time for sample evaluation.

[0097] In the above-mentioned sample evaluating apparatus, by contrast,even when only fluorometry is repeatedly executed with respect toindividual parts on the sample after initially measuring the pumpinglight time waveform, influences of time waveform shifts generated duringthe fluorometry can be minimized by employing a data analysis effectedby a plurality of fitting calculations. Therefore, evaluations ofindividual parts on the sample can be executed in a short timeaccurately and efficiently. In particular, since necessary conditionssuch as the fitting range, the initial position for starting a pluralityof fitting calculations, and the like are set beforehand, all of thefluorometry, data analysis, and sample evaluation for individual partson the measurement sample S can be executed automatically.

[0098] Without being restricted to the above-mentioned embodiments, thefluorescence measuring method and apparatus in accordance with thepresent invention can be modified in various manners. For example, it ispreferred that the condensing optical system 3, wavelength selectingmeans (spectroscope 4), and the like in the apparatus configurationshown in FIG. 1 have their favorable configurations according to thekind of the sample S to be measured, the wavelength of fluorescence, thepositional relationship between the pumping light source 1 andphotodetector 5, and the like. Though the driving control unit and dataprocessing unit are provided in the same controller 6 in the embodimentsshown in FIGS. 1 and 7, they may be disposed as separate drivingcontroller and data processor as well.

[0099] As for the measurement of pumping light and fluorescence, thepumping light may be measured after fluorometry, and the above-mentioneddata analysis may be carried out for each of their respective timewaveforms. Though fluorometry is initially carried out with thereference sample in the flowchart of FIG. 8, setting of a fitting rangeand the like can be carried out from the fluorescence time waveformobtained by the initial fluorometric operation in the measurement samplewithout using the reference sample.

[0100] As for the arrangement and movement of fluorescence and pumpinglight time waveforms on the time axis, various data analysis methods canbe employed without being restricted to the above-mentioned methodexecuting the fitting calculations while moving the fluorescence timewaveform from the initial position to the end position with the pumpinglight time waveform being arranged at a fixed time position on the timeaxis.

[0101] Namely, more generally, pumping light and fluorescence timewaveforms are arranged on the time axis such that a fluorescence peak ofthe fluorescence time waveform is placed at an initial position earlieror later by a predetermined time width than a time positionsubstantially coinciding with a pumping light peak of the pumping lighttime waveform, and then, while moving the fluorescence time waveform andfitting range or the pumping light time waveform with respect to thetime axis to an end position on the time axis on the opposite side ofthe time position where the fluorescence peak substantially coincideswith the pumping light peak from the initial position, wherebyfluorescence measuring method and apparatus which can compute waveformdata, individual physical quantities, and the like accurately andefficiency regardless of whether a shift occurs in the fluorescence timewaveform or not are obtained.

[0102] For example, in the above-mentioned embodiments, as shown inFIGS. 4A to 4D, the fluorescence time waveform F is disposed at theinitial position t1 earlier than the pumping light peak (FIG. 4C), andthen fitting calculations are carried out while moving the fluorescencetime waveform F to the end position t2 (FIG. 4D) later than the pumpinglight peak. Another data analysis method may be such that thefluorescence time waveform moves from an initial position located at atime position later than the pumping light peak such as the one shown inFIG. 4D to an end position earlier than the pumping light peak.Alternatively, while arranging the fluorescence time waveform F fixedlywith respect to the time axis, the pumping light time waveform E can bemoved from the initial position to the end position in a similar manner.

[0103] However, for simplifying the data analysis procedure, it ispreferred that, while one of the pumping light time waveform E and thefluorescence time waveform F is disposed at a fixed position on the timeaxis, the other be moved with respect to the time axis.

[0104] Also, the sample evaluating apparatus can similarly be modifiedin various manners. For example, in the flowchart shown in FIG. 8, iffluorescence fades in the sample when the fluorescence time waveform ismeasured (S206), the data analysis (S207) and sample evaluation (S208)may be skipped.

[0105] Though the quality evaluation of a semiconductor wafer isexplained by way of example as a sample to be evaluated, the presentinvention can be employed for various other samples as well. Forexample, fluorescence lifetime measurement, time-resolved fluorescenceanisotropy measurement, and the like have been considered effectiveevaluating means in mask screening, drug screening, and the like carriedout while using a microtiter plate or the like as a sample in theprocess of developing drugs. Automatic evaluations with a highthroughput are required in this case as in the semiconductor wafer,whereby employing the sample evaluating apparatus of FIG. 7, theflowchart of FIG. 8, and the like enables efficient sample evaluations.

[0106] Industrial Applicability

[0107] The fluorescence measuring method and apparatus in accordancewith the present invention, and a sample evaluating apparatus using thesame can be utilized as fluorescence measuring method and apparatuswhich can compute waveform data and physical quantities such asfluorescence lifetime efficiently with a sufficient accuracy regardlessof whether a shift occurs in the fluorescence time waveform or not, anda sample evaluating apparatus using the same.

[0108] Namely, the pumping light and fluorescence time waveforms arearranged at their predetermined time positions with respect to the timeaxis used for a data analysis for determining a fluorescence lifetime orthe like, and a plurality of fitting calculations are carried out whilemoving the fluorescence time waveform and fitting range or the pumpinglight time waveform from the initial position where the fluorescencepeak is earlier or later by a predetermined time width than the pumpinglight peak to the end position on the opposite side. Then, from thusdetermined plurality of waveform data items, an optimal measurementwaveform data is selected according to a selection criterion such as theχ² value obtained by a fitting calculation. This makes it possible tocompute waveform data and physical quantities such as fluorescencelifetime efficiently with a sufficient accuracy regardless of whether ashift occurs in the fluorescence time waveform or not.

[0109] Such fluorescence measuring apparatus and sample evaluatingapparatus can greatly shorten the time required for measurement andevaluation, in particular, when carrying out a number of fluorometricoperations in semiconductor wafer quality evaluations, screening fordeveloping drugs, and the like. Further, since measurement andevaluation become efficient as such, applications are expected to becomepossible in a wide range with respect to sample evaluations other thanthe semiconductor wafer evaluation and screening for developing drugs aswell.

1. A fluorescence measuring method comprising: a pumping step ofirradiating a sample with pulsed pumping light; a light-detecting stepof detecting fluorescence released from said sample pumped with saidpumping light; and a data processing step of subjecting a time waveformof fluorescence detected by said light-detecting step to a data analysisincluding a fitting calculation in a fitting range acting as apredetermined time range fixedly set for said time waveform offluorescence so as to compute waveform data; wherein, in said dataprocessing step, a time waveform of pumping light determined beforehandand said time waveform of fluorescence are arranged on a time axis usedfor said fitting calculation such that a fluorescence peak of said timewaveform of fluorescence is placed at an initial position earlier orlater by a predetermined time width than a time position substantiallycoinciding with a pumping light peak of said time waveform of pumpinglight, and then, while moving said time waveform of fluorescence andfitting range or said time waveform of pumping light with respect tosaid time axis to an end position on said time axis on the opposite sideof said time position where said fluorescence peak substantiallycoincides with said pumping light peak from said initial position,respective fitting calculations are carried out at a plurality of timepositions different from each other with reference to said time waveformof pumping light, and a waveform data item selected according to apredetermined selection criterion from a plurality of waveform dataitems respectively computed in said fitting calculations is employed asfinal measurement waveform data.
 2. A fluorescence measuring methodaccording to claim 1, wherein, in said data processing step, one of saidtime waveform of pumping light and said time waveform of fluorescence isarranged at a fixed time position on said time axis, and the other ismoved with respect to said time axis.
 3. A fluorescence measuring methodaccording to claim 1, wherein, in said data processing step, afluorescence lifetime is determined according to said measurementwaveform data.
 4. A fluorescence measuring method according to claim 1,wherein, in said data processing step, χ² values respectively determinedin said fitting calculations are used as said selection criterion, andsaid waveform data computed in said fitting calculation yielding theminimal χ² value is selected as said measurement waveform data.
 5. Afluorescence measuring method according to claim 1, wherein, in saiddata processing step, a time range set with reference to saidfluorescence peak of said time waveform of fluorescence is used as saidfitting range.
 6. A fluorescence measuring method according to claim 1,further comprising a range setting step of setting said fitting rangeprior to said pumping step.
 7. A fluorescence measuring apparatuscomprising: pumping means for irradiating a sample with pulsed pumpinglight; light-detecting means for detecting fluorescence released fromsaid sample pumped with said pumping light; and data processing meansfor subjecting a time waveform of fluorescence detected by saidlight-detecting means to a data analysis including a fitting calculationin a fitting range acting as a predetermined time range fixedly set forsaid time waveform of fluorescence so as to compute waveform data;wherein said data processing means arranges a time waveform of pumpinglight determined beforehand and said time waveform of fluorescence on atime axis used for said fitting calculation such that a fluorescencepeak of said time waveform of fluorescence is placed at an initialposition earlier or later by a predetermined time width than a timeposition substantially coinciding with a pumping light peak of said timewaveform of pumping light, and then, while moving said time waveform offluorescence and fitting range or said time waveform of pumping lightwith respect to said time axis to an end position on said time axis onthe opposite side of said time position where said fluorescence peaksubstantially coincides with said pumping light peak from said initialposition, carries out respective fitting calculations at a plurality oftime positions different from each other with reference to said timewaveform of pumping light, and employs a waveform data item selectedaccording to a predetermined selection criterion from a plurality ofwaveform data items respectively computed in said fitting calculationsas final measurement waveform data.
 8. A fluorescence measuringapparatus according to claim 7, wherein said data processing meansarranges one of said time waveform of pumping light and said timewaveform of fluorescence at a fixed time position on said time axis andmoves the other with respect to said time axis.
 9. A fluorescencemeasuring apparatus according to claim 7, wherein said data processingmeans determines a fluorescence lifetime according to said measurementwaveform data.
 10. A fluorescence measuring apparatus according to claim7, wherein said data processing means uses χ² values respectivelydetermined in said fitting calculations as said selection criterion, andselects said waveform data computed in said fitting calculation yieldingthe minimal χ² value as said measurement waveform data.
 11. Afluorescence measuring apparatus according to claim 7, wherein said dataprocessing means uses a time range set with reference to saidfluorescence peak of said time waveform of fluorescence as said fittingrange.
 12. A fluorescence measuring apparatus according to claim 7,further comprising range setting means for setting said fitting rangebeforehand.
 13. A sample evaluating apparatus comprising: thefluorescence measuring apparatus according to claim 7; and sampleevaluating means for evaluating said sample by comparing saidmeasurement waveform data obtained in said data processing means of saidfluorescence measuring apparatus and reference waveform data determinedbeforehand with each other.